The present invention relates to monomers and oligomers containing guanidinium moieties and methods of preparing such oligomers. The oligomers of the present invention are used for investigative and therapeutic purposes.
It is well known that most of the bodily states in mammals, including most disease states, are affected by proteins. Classical therapeutic modes have generally focused on interactions with such proteins in an effort to moderate their disease-causing or disease-potentiating functions. However, recently, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, maximum therapeutic effect and minimal side effects may be realized. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides. Oligonucleotides are now accepted as therapeutic agents with great promise. Oligonucleotides are known to hybridize to single-stranded DNA or RNA molecules. Hybridization is the sequence-specific base pair hydrogen bonding of nucleobases of the oligonucleotide to the nucleobases of the target DNA or RNA molecule. Such nucleobase pairs are said to be complementary to one another. The concept of inhibiting gene expression through the use of sequence-specific binding of oligonucleotides to target RNA sequences, also known as antisense inhibition, has been demonstrated in a variety of systems, including living cells (for example see: Wagner et al., Science (1993) 260: 1510-1513; Milligan et al., J. Med. Chem., (1993) 36:1923-37; Uhlmann et al., Chem. Reviews, (1990) 90:543-584; Stein et al., Cancer Res., (1988) 48:2659-2668).
The events that provide the disruption of the nucleic acid function by antisense oligonucleotides (Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, (1989) CRC Press, Inc., Boca Raton, Fla.) are thought to be of two types. The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides: Miller and Ts""O, Anti-Cancer Drug Design, 1987, 2:117-128, and xcex1-anomer oligonucleotides are the two most extensively studied antisense agents which are thought to disrupt nucleic acid function by hybridization arrest.
The second type of terminating event for antisense oligonucleotides involves the enzymatic cleavage of the targeted RNA by intracellular RNase H. A 2xe2x80x2-deoxyribofuranosyl oligonucleotide or oligonucleotide analog hybridizes with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate oligonucleotides are the most prominent example of an antisense agent that operates by this type of antisense terminating event.
Oligonucleotides may also bind to duplex nucleic acids to form triplex complexes in a sequence specific manner via Hoogsteen base pairing (Beal et al., Science, (1991) 251:1360-1363; Young et al., Proc. Natl. Acad. Sci. (1991) 88:10023-10026). Both antisense and triple helix therapeutic strategies are directed towards nucleic acid sequences that are involved in or responsible for establishing or maintaining disease conditions. Such target nucleic acid sequences may be found in the genomes of pathogenic organisms including bacteria, yeasts, fungi, protozoa, parasites, viruses, or may be endogenous in nature. By hybridizing to and modifying the expression of a gene important for the establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.
In determining the extent of hybridization of an oligonucleotide to a complementary nucleic acid, the relative ability of an oligonucleotide to bind to the complementary nucleic acid may be compared by determining the melting temperature of a particular hybridization complex. The melting temperature (Tm), a characteristic physical property of double helices, denotes the temperature (in degrees centigrade) at which 50% helical (hybridized) versus coil (unhybridized) forms are present. Tm is measured by using the UV spectrum to determine the formation and breakdown (melting) of the hybridization complex. Base stacking, which occurs during hybridization, is accompanied by a reduction in UV absorption (hypochromicity). Consequently, a reduction in UV absorption indicates a higher Tm. The higher the Tm, the greater the strength of the bonds between the strands.
Oligonucleotides may also be of therapeutic value when they bind to non-nucleic acid biomolecules such as intracellular or extracellular polypeptides, proteins, or enzymes. Such oligonucleotides are often referred to as xe2x80x98aptamersxe2x80x99 and they typically bind to and interfere with the function of protein targets (Griffin et al., Blood, (1993), 81:3271-3276; Bock et al., Nature, (1992) 355: 564-566).
Oligonucleotides and their analogs have been developed and used for diagnostic purposes, therapeutic applications and as research reagents. For use as therapeutics, oligonucleotides must be transported across cell membranes or be taken up by cells, and appropriately hybridize to target DNA or RNA. These critical functions depend on the initial stability of the oligonucleotides toward nuclease degradation. A serious deficiency of unmodified oligonucleotides which affects their hybridization potential with target DNA or RNA for therapeutic purposes is the enzymatic degradation of administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes referred to as nucleases. For oligonucleotides to be useful as therapeutics or diagnostics, the oligonucleotides should demonstrate enhanced binding affinity to complementary target nucleic acids, and preferably be reasonably stable to nucleases and resist degradation. For a non-cellular use such as a research reagent, oligonucleotides need not necessarily possess nuclease stability.
A number of chemical modifications have been introduced into oligonucleotides to increase their binding affinity to target DNA or RNA and resist nuclease degradation.
Modifications have been made to the ribose phosphate backbone to increase the resistance to nucleases. These modifications include use of linkages such as methyl phosphonates, phosphorothioates and phosphorodithioates, and the use of modified sugar moieties such as 2xe2x80x2-O-alkyl ribose. Other oligonucleotide modifications include those made to modulate uptake and cellular distribution. A number of modifications that dramatically alter the nature of the internucleotide linkage have also been reported in the literature. These include non-phosphorus linkages, peptide nucleic acids (PNA""s) and 2xe2x80x2-5xe2x80x2 linkages. Another modification to oligonucleotides, usually for diagnostic and research applications, is labeling with non-isotopic labels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules.
A variety of modified phosphorus-containing linkages have been studied as replacements for the natural, readily cleaved phosphodiester linkage in oligonucleotides. In general, most of them, such as the phosphorothioate, phosphoramidates, phosphonates and phosphorodithioates all result in oligonucleotides with reduced binding to complementary targets and decreased hybrid stability. In order to make effective therapeutics therefore this binding and hybrid stability of antisense oligonucleotides needs to be improved.
Of the large number of modifications made and studied, few have progressed far enough through discovery and development to deserve clinical evaluation. Reasons underlying this include difficulty of synthesis, poor binding to target nucleic acids, lack of specificity for the target nucleic acid, poor in vitro and in vivo stability to nucleases, and poor pharmacokinetics. Several phosphorothioate oligonucleotides and derivatives are presently being used as antisense agents in human clinical trials for the treatment of various disease states. A submission for approval was recently made to both United States and European regulatory agencies for one antisense drug, Fomivirsen, for use to treat cytomegalovirus (CMV) retinitis in humans.
The structure and stability of chemically modified nucleic acids is of great importance to the design of antisense oligonucleotides. Over the last ten years, a variety of synthetic modifications have been proposed to increase nuclease resistance, or to enhance the affinity of the antisense strand for its target mRNA (Crooke et al., Med. Res. Rev., 1996, 16, 319-344; De Mesmaeker et al., Acc. Chem. Res., 1995, 28, 366-374). Although a great deal of information has been collected about the types of modifications that improve duplex formation, little is known about the structural basis for the improved affinity observed.
RNA exists in what has been termed xe2x80x9cA Formxe2x80x9d geometry while DNA exists in xe2x80x9cB Formxe2x80x9d geometry. In general, RNA:RNA duplexes are more stable, or have higher melting temperatures (Tm) than DNA:DNA duplexes (Sanger et al., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stability of RNA has been attributed to several structural features, most notably the improved base stacking interactions that result from an A-form geometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2xe2x80x2 hydroxyl in RNA biases the sugar toward a C3xe2x80x2 endo pucker, i.e., also designated as Northern pucker, which causes the duplex to favor the A-form geometry. On the other hand, deoxy nucleic acids prefer a C2xe2x80x2 endo sugar pucker, i.e., also known as Southern pucker, which is thought to impart a less stable B-form geometry (Sanger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York, N.Y.). In addition, the 2xe2x80x2 hydroxyl groups of RNA can form a network of water mediated hydrogen bonds that help stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35, 8489-8494).
DNA:RNA hybrid duplexes, however, are usually less stable than pure RNA:RNA duplexes, and depending on their sequence may be either more or less stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid duplex is intermediate between A- and B-form geometries, which may result in poor stacking interactions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of a DNA:RNA hybrid is central to antisense therapies as the mechanism requires the binding of a modified DNA strand to a mRNA strand. To effectively inhibit the mRNA, the antisense DNA should have a very high binding affinity with the mRNA. Otherwise the desired interaction between the DNA and target mRNA strand will occur infrequently, thereby decreasing the efficacy of the antisense oligonucleotide.
One synthetic 2xe2x80x2-modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2xe2x80x2-methoxyethoxy (MOE, 2xe2x80x2xe2x80x94OCH2CH2OCH3) side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25, 4429-4443). One of the immediate advantages of the MOE substitution is the improvement in binding affinity, which is greater than many similar 2xe2x80x2 modifications such as O-methyl, O-propyl, and O-aminopropyl (Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443). 2xe2x80x2-O-Methoxyethyl-substituted also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926). Relative to DNA, they display improved RNA affinity and higher nuclease resistance. Chimeric oligonucleotides with 2xe2x80x2-O-methoxyethyl-ribonucleoside wings and a central DNA-phosphorothioate window also have been shown to effectively reduce the growth of tumors in animal models at low doses. MOE substituted oligonucleotides have shown outstanding promise as antisense agents in several disease states. One such MOE-substituted oligonucleotide is currently available for the treatment of CMV retinitis.
Although the known modifications to oligonucleotides, including the use of the 21-O-methoxyethyl modification, have contributed to the development of oligonuclotides for use in diagnostics, therapeutics and as research reagents, there still exists a need in the art for further modifications to oligonucleotides having enhanced hybrid binding affinity and/or increased nuclease resistance.
In accordance with the present invention, oligomers containing guanidinium groups are provided. The present invention provides monomers of the formula: 
wherein:
Bx is a heterocyclic base;
T1 is OH or a protected hydroxyl group;
T2 is an activated phosphorus group or a linking moiety attached to a solid support;
T3 is H, OH, a protected hydroxyl or a sugar substituent group; said monomer further comprising at least one group, R1, therein; said R1 group occurring in lieu of at least one T1, T2 or T3 or as a substituent on at least one Bx; said R1 group having the formula: 
xe2x80x83wherein:
each Z is, independently, a single bond, O, N or S;
each R2, R3, R3xe2x80x2, and R4 is, independently, hydrogen, C(O)R5, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;
or R3 and R4, together, are R7;
each R5 is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
each R7 is, independently, hydrogen or forms a phthalimide moiety with the nitrogen atom to which it is attached;
each m is, independently, zero or 1; and
each n is, independently, an integer from 1 to about 6.
Preferred compositions include oligomers comprising a plurality of nucleotide units of the structure: 
wherein:
Bx is a heterocyclic base;
each T1 and T2 is, independently, OH, a protected hydroxyl, a nucleotide, a nucleoside or an oligonucleotide;
T3 is H, OH, a protected hydroxyl or a sugar substituent group; said oligomer further comprising at least one group, R1, therein; said R1 group occurring at the 3xe2x80x2 end, the 5xe2x80x2 end, in lieu of at least one T3 or as a substituent on at least one Bx; said R1 group having the formula: 
xe2x80x83wherein:
each Z is, independently, a single bond, O, N or S;
each R2, R3, R3xe2x80x2, and R4 is, independently, hydrogen, C(O)R5, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;
or R3 and R4, together, are R7;
each R5 is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
each R7 is, independently, hydrogen or forms a phthalimide moiety with the nitrogen atom to which it is attached;
each m is, independently, zero or 1; and
each n is, independently, an integer from 1 to about 6.
In a preferred embodiment, R1, R2, R3, R3xe2x80x2, and R4 are hydrogen. In another preferred embodiment, R1, R2, R3, R3xe2x80x2, and R4 are hydrogen, m is zero and n is 2.
The present invention also provides methods for preparing oligomers comprising the steps of:
(a) selecting a monomer of the formula: 
xe2x80x83wherein:
Bx is a heterocyclic base;
T1 is OH or a protected hydroxyl group;
T2 is a linking moiety attached to a solid support;
T3 is H, OH, a protected hydroxyl or a sugar substituent group; said monomer further comprising at least one group, R1, therein; said R1 group occurring in lieu of at least one T1, T2 or T3 or as a substituent on at least one Bx; provided that if T2 is R1, T3 is a linking moiety attached to a solid support; said R1 group having the formula: 
xe2x80x83wherein:
each Z is, independently, a single bond, O, N or S;
each R2, R3, R3xe2x80x2, and R4 is, independently, hydrogen, C(O)R5, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl,aryl, alkenyl and alkynyl;
or R3 and R4, together, are R7;
each R5 is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
each R7 is, independently, hydrogen or forms a phthalimide moiety with the nitrogen atom to which it is attached;
each m is, independently, zero or 1; and
each n is, independently, an integer from 1 to about 6;
(b) deprotecting the protected hydroxyl group at the 5xe2x80x2-position to form a deprotected monomer;
(c) coupling said deprotected monomer with a second monomer of formula: 
xe2x80x83wherein:
Bx is a heterocyclic base;
T1 is OH or a protected hydroxyl group;
T2 is an activated phosphorus group;
T3 is H, OH, a protected hydroxyl or a sugar substituent group; said monomer further comprising at least one group, R1, therein; said R1 group occurring in lieu of at least one T1, T2 or T3 or as a substituent on at least one Bx; provided that if T2 is R1, T3 is an activated phosphorus group; said R1 group having the formula: 
xe2x80x83wherein:
each Z is, independently, a single bond, O, N or S;
each R2, R3, R3xe2x80x2, and R4 is, independently, hydrogen, C(O)R5, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;
or R3 and R4, together, are R7;
each R5 is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
each R7 is, independently, hydrogen or forms a phthalimide moiety with the nitrogen atom to which it is attached;
each m is, independently, zero or 1; and
each n is, independently, an integer from 1 to about 6, said coupling occurring in the presence of an activating agent to form a coupled compound;
(d) capping said coupled compound with a capping reagent to form a capped compound having an interncleotide linkage;
(e) oxidizing said interncleotide linkage with an oxidizing reagent; and
(f) repeating steps (b) to (e) to form an oligomer. In one embodiment of the present invention, the method includes an additional step of cleaving the oligomer with a cleaving reagent.
Preferred activating reagents include tetrazole, pyridinium trifluoroacetate and dicyanoimidazole. It is preferred that acetic anhydride and N-methylimidazole be the capping reagent. Preferred oxidizing reagents include iodine, camphorsulfonyloxaziridine, t-butyl hydrogen peroxide and Beaucage reagent.
The present invention also provides compounds of the formula: 
wherein X is cyanoethyloxy, benzyloxy, t-butoxy, methoxy, ethoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, trifluoromethyl, butyryl, iso-butyryl, phenyl or aryl.
In one embodiment of the present invention, X is cyanoethyloxy. In another embodiment, X is phenyl. In yet another embodiment, X is t-butyl.
The present invention is also directed to non-nucleic acid monomers and oligomers comprising at least one such non-nucleic acid monomer. The present invention includes non-nucleic acid monomers of the formula: 
wherein:
X is C3-C10 alkyl, C6-C24 aryl, C6-C24 heteroaryl, C4-C20 alicyclic, C4-C20 alicyclic having at least one heteroatom, nucleoside, nucleotide or oligonucleotide;
Y1 is a hydroxyl protecting group;
Y2 is an activated phosphorus group or a linking moiety attached to a solid support;
each R2, R3, R3xe2x80x2, and R4 is, independently, hydrogen, C(O)R5, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, substituted or unsubstituted C2-C10 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugate group, wherein the substituent groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;
or R3 and R4, together, are R7;
each R5 is, independently, substituted or unsubstituted C1-C10 alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl, iso-butyryl, phenyl or aryl;
each R7 is, independently, hydrogen or forms a phthalimide moiety with the nitrogen atom to which it is attached; and
n is an integer from 1 to about 6.
In a preferred embodiment, Y1 is dimethoxytrityl. In another preferred embodiment, Y1 is monomethoxytrityl. In yet another preferred embodiment, Y2 is a phosphoramidite. In a further embodiment, Y2 is a linking moiety attached to a solid support. It is preferred that Y2 be succinyl CPG.